The present invention relates to a novel splicing variants of presenilin-2 along with a method for screening and a method for identifying therapeutic agents for central nervous system diseases. The present invention also relates to a method for examining central nervous system diseases.
In the genome DNA of eucaryotes, sequences corresponding to mature mRNA are frequently present separated at several locations. In such cases, gene transcription is performed continuously over the entire region, including those sequences corresponding to mature mRNA, resulting in the production of precursor mRNA (pre-mRNA) (transcription products also containing portions not required by mature mRNA). This precursor mRNA is then subjected to processing to become mature mRNA. During the course of processing, simultaneous to the addition of a cap structure and poly A, those portions not required by the mRNA (introns or intervening sequences) are cut out, while the portions corresponding to the mature mRNA (exons) are joined to form mature mRNA. These cuttting and joining processes are referred to as xe2x80x9csplicingxe2x80x9d. Splicing is a complicated and delicate process in which a plurality of severing and coupling processes are regulated by the involvement of a large protein-RNA aggregate referred to as a spliceosome. For example, although different types of mature mRNA, namely xe2x80x9csplicing variantsxe2x80x9d, are frequently formed due to a mutation and so forth that occurs in the boundary region between an intron and exon on the genome, some of these variants are known to give rise to functionally abnormal mutant proteins that are capable of causing disease. Research has been conducted thus far on splicing variants in causative genes in order to elucidate the mechanism of pathogenesis for various diseases and disorders, and splicing variants have also been used as diagnostic markers for certain diseases.
On the other hand, the amyloid precursor protein (APP) gene on chromosome 21, the presenilin-1 (PS-1) gene on chromosome 14 and the presenilin-2 (PS-2) gene on chromosome 1 have been previously determined to be the major causative genes of familial Alzheimer""s disease (FAD). Subsequently, research has been conducted while focusing on mutations and splicing variants and so forth discovered in these causative genes on the relationship between these and the mechanism of pathogenesis of Alzheimer""s disease. However, very little has been determined for the mechanism of pathogenesis of sporadic Alzheimer""s disease, which constitutes the majority of Alzheimer""s disease.
For example, mRNA originating in the brain tissue of familiar or sporadic Alzheimer""s disease patients and normal subjects was analyzed for presenilin-1 gene (Sherrington et al., Nature, Vol. 375, pp. 754-760, 1995), and the existence of various splicing variants has been reported (Clark et al,. Nature Genetics, Vol. 11, pp. 219-222, 1995; Anwar et al., Journal of Neurochemistry, Vol. 66, pp. 1774-1777, 1996).
Similar to presenilin-1 gene, presenelin-2 gene (Levy-Lahad et al., Genomics, Vol. 34, pp. 198-204, 1996; Levy-Lahad et al., Science, Vol. 269, pp. 973-977, 1995; Rogaev et al., Nature, Vol. 376, pp. 775-778, 1995) is composed of 12 exons, and 10 of these exons (exons 3-12) are known to code proteins. In addition, the existence of splicing variants lacking exon 8 as well as splicing variants simultaneously lacking exon 3 and exon 4 has been reported in normal human tissue (Prihar et al., NeuroReport, Vol. 7, pp. 1680-1684, 1996). However, there have been no splicing variants specific to Alzheimer""s disease (and particularly sporadic Alzheimer""s disease) reported for presenilin-2 gene.
An object of the present invention is to provide a novel method for screening and a novel method for identifying therapeutic agents for central nervous system diseases (such as Alzheimer""s disease). In addition, an object of the present invention is to provide a novel method for examining central nervous system diseases (such as Alzheimer""s disease). In addition, another object of the present invention is to provide a novel splicing variant originating in presenilin-2 that is useful in research on central nervous system diseases.
The present inventors found that expression of abnormal presenilin-2 mRNA splicing variants not found in the normal state ((i) splicing variant lacking exon 5, (ii) splicing variant lacking exon 3, and (iii) splicing variant lacking both exon 3 and exon 4 and retaining a portion of an intron sequence) is induced by exposure to oxidative stress or xcex2-amyloid stimulation in the culture system of nerve cells. In addition, when the expression in human brain tissue was investigated for one of these splicing variants that is lacking exon 5, the present inventors made the unique discovery that this splicing variant lacking exon 5 is present at high frequency in the brain tissue of sporadic Alzheimer""s disease patients, thereby leading to completion of the present invention.
Namely, the present invention is a method for screening therapeutic agents or preventive agents for central nervous system diseases, and a method for identifying the same, which comprises assaying the suppressing effect of a test substance on the expression of a splicing variant transcribed from presenilin-2 gene.
In addition, the present invention is a method for examining central nervous system diseases which comprises detecting the expression of a splicing variant transcribed from presenilin-2 gene in a test sample originated from an animal individual.
Moreover, the present invention is a nucleic acid having a base sequence originating in a splicing variant transcribed from presenilin-2 gene (in the following, referred to as a presenilin-2 splicing variant) and a polypeptide coded by said splicing variant.